MicroRNAs as potential indicators of the development and progression of uterine leiomyoma

Recent studies demonstrated a significant role of several microRNAs (miRs) in the development of leiomyoma. Here, we investigated miR expression profiles using microarray and found a significantly higher expression of miRs in leiomyoma than in adjacent myometrium. We also confirmed the upregulation of five selected miRs including miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p in cellular proliferation, extracellular matrix turnover, and angiogenesis by RT-qPCR. Interestingly, the miRs showed a higher expression in cases of large leiomyoma or in patients with a history of transfusion due to anemia. We then analyzed the expression of the miR target molecules including Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Insulin-like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1) via immunohistochemistry. TGFBR2 and IGF2BP1 were positively stained in 81% and 62.5% of leiomyoma tissues but not in adjacent myometrium. Both were more frequently positive in patients with ≥ 6 cm leiomyoma and mass effect. The mean expression levels of miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p were higher in cases with TGFBR2 and IGF2BP1 positive leiomyoma. We observed several miRs were overexpressed in leiomyoma tissues, and these results provide insight into the role of miRs in the development and progression of leiomyoma and underscore the need to validate their utility as diagnostic or therapeutic targets.


Introduction
Uterine leiomyoma is the most common benign tumor in women of reproductive age and represent an important public health issue. Patients usually present with abnormal uterine bleeding and sometimes complain of abdominal discomfort due to compression by the mass. The manifestations change throughout lifetime such as continuously growing or decreasing mass, irregular bleeding and rarely malignant transformation. Several treatment options exist ranging from observation or medical therapy including iron supplementation, oral contraceptive, and progesterone, uterine artery embolization and focused ultrasound, hysteroscopy or laparoscopic myomectomy to hysterectomy. Therefore, precise diagnosis, regular follow-up, and adequate treatment are important especially when patients show symptomatic changes related to uterine leiomyoma [1]. Clinicians should evaluate not only the symptoms but also associated conditions such as anemia, fertility preservation, and the possibility of malignancy before performing hysterectomy. Although uterine leiomyoma is the most common cause of hysterectomy, its pathogenesis remains unclear, and hence the limited number of treatment options available for symptomatic patients. Recent studies compared the microRNA (miR) expression of uterine leiomyoma and myometrium and reported that a few specific miRs such as miR -150-5P [2], miR-29 family [3], and miR-let-7 [4] played a pathogenic role in the development of leiomyoma. miR expression profiles significantly differ among organs and tissues, and one miR has numerous targets. Although miR levels in cancer tissues are generally lower than those in normal tissues [5]. However, their clinical impact, specifically on disease severity, is not fully understood.
In our study, we analyzed miR in a sample set of 19 patients with leiomyoma using microarrays and real-time quantitative PCR (RT-qPCR). We observed distinct miR expression profiles between leiomyoma and adjacent myometrium and selected miRs showing prominent differences. We then selected two target molecules of selected miRs, Transforming Growth Factor Beta Receptor 2 (TGFBR2) and Insulin-like Growth Factor 2 mRNA Binding Protein 1 (IGF2BP1), for immunohistochemical analysis of corresponding tissue samples. The reported findings validate the role of miRs in mediating changes associated with the development and severity of leiomyoma.

Study subjects and tissue specimens
The study enrolled 19 women who received surgical management for uterine leiomyoma (heavy menstrual bleeding, palpable mass) from May, 2018 to February, 2019 in a university hospital. The study received institutional review board approval from Bucheon St. Mary's Hospital, The Catholic University of Korea (HCI8SESI0037), and all participants provided written informed consent. We conducted this study under the amended Declaration of Helsinki on the ethical conduct of research involving human subjects. Clinical data were analyzed through chart review, and the association between miR and cytokine was investigated.
Uterine tissues were collected from leiomyoma mass and adjacent (within 2 cm) myometrial tissues within 1 hour of surgery after examine by gynecologist and pathologist. miR was extracted from RNA in later-preserved tissues using a miRNeasy Mini Kit (Qiagen, Hilden, Germany) at -80˚C, following the manufacturer's protocol. RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

miR profiling using array
The miRs of 10 leiomyoma masses and 6 myometrial tissues were profiled using the Affymetrix Genechip miR 4.0 array according to the manufacturer's protocol. RNA samples (130 ng) were labeled with the FlashTag™ Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA). The labeled RNA was quantified, fractionated and hybridized to the miR microarray according to the standard procedures provided by Macrogen Inc. (Seoul, Korea). The labeled RNA was heated to 99˚C for 5 min and then to 45˚C for 5 min. RNA-array hybridization was performed via agitation at 60 rotations per min for 16 h at 48˚C on an Affymetrix Hybridization oven. The chips were washed and stained using a Genechip Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA). The chips were then scanned with an Affymetrix GCS 3000 scanner (Affymetrix). Signal values were computed using the Affymetrix 1 GeneChip™ Command Console software. Raw data were extracted automatically via Affymetrix data extraction protocol using the software provided by Affymetrix GeneChip 1 Command Console 1 Software (AGCC). The CEL files were imported and miR level RMA+DABG were analyzed. All analyses and results were exported using Affymetrix 1 Power Tools (APT) Software. After normalization, significant differentially expressed miRs were identified through Volcano Plot filtering between the two experimental groups. Finally, hierarchical clustering was performed for distinct miR expression profiling with at least 3.0-fold expression and P <0.05 of the samples.

Immunohistochemistry (IHC) of TGFBR2 and IGF2BP1
IHC was performed to evaluate the expression of TGFBR2 and IGF2BP1 in 16 leiomyoma tissues. Tissues were fixed in formalin and embedded in paraffin, and 3-μm-thick paraffin sections were obtained. IHC was performed using an automated immunohistochemical stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA) according to the manufacturer's protocol. The sections were deparaffinized and pretreated with a cell-conditioning solution (CC1, Ventana), followed by UV irradiation to abrogate the endogenous hydroperoxidase activity. The primary antibodies were diluted in Dako antibody diluent (Dako Cytomation, Glostrup, Denmark) with background-reducing components and used under the following dilutions: TGFBR2 (1:100, ab61213, Abcam, Franklin Lakes, NJ, USA) and IGF2BP1 (1:100, ab82968, Abcam,). The sections were incubated with primary antibodies at room temperature for 32 min, and hybridized with HRP-conjugated secondary antibody (Ventana) for 8 min. The reaction was developed with diaminobenzidine (DAB; Dako) for 5 min and the slides were counterstained with hematoxylin II (Ventana) for 4 min and bluing reagent (Ventana) for 4 min. The sections were observed under light microscope (BX50, Olympus, Japan).

Statistical analysis
The comparative analysis of test and control samples was carried out using an independent ttest and fold change was determined, based on the null hypothesis stating that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting the P value using Benjamini-Hochberg algorithm. All statistical tests and visualization of differentially expressed genes were conducted using R statistical language 3.3.3.
In real-time PCR, statistical analysis of the association between miR expression and different tumor histotypes was conducted using the non-parametric Mann-Whitney test. The Tukey's multiple comparisons test was used to evaluate the differences in miR expression according to clinicopathological parameters. Pearson's chi-square test was utilized to analyze the relationship between parametric data. Pearson's correlation was performed for the quantitative analysis of size of leiomyoma and miR expression. All statistical analyses were performed using SPSS Statistics for Windows, Version 20.0 (IBM Corp., Amonk, NY, USA). P <0.05 was considered to indicate a statistically significant difference.

Patient's characteristics
The clinical characteristics of 19 participants are summarized in Table 1. The median age was 46 years and BMI was 22.9 ± 2.7 kg/m 2 . Irregular menstruation and heavy menstrual bleeding were reported by six (31.6%) and eight (42.1%) participants, respectively. Indications for surgery were mass effects including pressure symptoms and/or palpable mass (n = 8), heavy menstrual bleeding (n = 8) and both symptoms (n = 3). The mean size of the uterine leiomyoma was 7.3 ± 3.7 cm (2-15 cm) in its largest diameter. Eight patients (42.1%) received oral iron supplementation and three patients (15.8%) had a history of transfusion of packed red cells (PRC) due to anemia. Total laparoscopic hysterectomy was performed in 18 patients and myomectomy was conducted in 1 patient.

Clinical significance of TGFBR2 and IGF2BP1 expression
We screened the predictable target molecules of differentially expressed miRs via the TargetScan (https://www.targetscan.org/) and miRDB (http://mirdb.org) and selected TGFBR2 and IGF2BP1, which were recurrently nominated. We also reviewed previous studies [3,17,18] revealing the association of miRs and selected two target molecules. In addition, we confirmed their association using the Ingenuity 1 Pathway Analysis (IPA; QIAGEN Inc.) (Fig 4). IHC was successfully performed for two targets in 16 samples. We found that TGFBR2 and IGF2BP1 were positively stained in 13 (81%) and 10 (62.5%) leiomyoma tissues; however, neither of them was stained in the adjacent myometrium (S3 Fig). IGF2BP1 was more frequently positive in patients with � 6 cm leiomyoma, irregular menstruation and mass effect. TGFBR2 was more frequently positive in patients with � 6 cm leiomyoma and mass effect ( Table 2). The mean expression levels of miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p were higher in patients with IGF2BP1 and TGFBR2-positive leiomyoma. However, no statistical significant difference was found between the two groups (S4 Fig).
We measured the expression of 5 up-regulated miRs in leiomyoma including miR-181a-5p, 127-3p, 28-3p, 30b-5p, and let-7c-5p using RT-qPCR, and found their interaction with the size of leiomyoma. The expression of miR-181a-5p and 127-3p strongly up-regulated during myoblast differentiation [6,9,11]. miR-181a is recognized as a senescence-associated miR and a potent negative regulator of Akt pathway [24]. It also affected the expression of insulin-like growth factor binding proteins (IGFBP), which might be of some importance in leiomyomas [7]. miR-127-3p is expressed at significantly higher level in proliferating rather than

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differentiating cells [10]. The miR-28-3p is a crucial regulator of myogenic differentiation of rhabdomyosarcoma cells and myoblasts [12]. The expression of miR-30b-5p was associated with differentiation of both skeletal and vascular smooth muscles via interaction with MBNL1 and/or MBNL2 [13,14]. Let-7 is one of the most widely studied miRs and let-7c-5p is important for cell growth, proliferation and inflammation [25]. Genetic alterations, cellular transformation of myometrial cells, ovarian steroids may affect the expression of Let-7 family and their aberrant expression alter the stability of their target gens (vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), interleukin(IL),TGF, tumor necrosis factor(TNF) et al).The product of some of these genes may in turn regulate the expression of miR, which through a feedback mechanism influence various cellular activities including tissue turnover, cell-cycle progression, hypertrophy, angiogenesis, and inflammation resulting in tissue fibrosis, a characteristic of leiomyoma during growth [26]. Let-7 expression was significantly decreased in leiomyosarcoma tissues, which may contribute to the malignant transformation of leiomyoma accompanied by overexpression of HMGA2 [15,16].Therefore, we postulated that the up-regulated miRs modify target molecules in addition to proliferation and differentiation of muscle cells and were associated with the development and growth of leiomyoma.
Although the expression and possible regulatory functions on miRs in leiomyoma with particular emphasis on the expression of their selective target genes whose products effect various cellular activities critical to pathogenesis of leiomyomas, there are few studies on the definite evidence for the association. We investigated the expression of two target molecules of those miRs via IHC. Leiomyoma tissues but not the adjacent myometrium stained positive for TGFBR2 and IGF2BP1. TGFBR2 is a receptor of a secreted polypeptide TGF-beta2, which regulates leiomyoma growth [27].The IGF2BP1, an oncofetal RNA-binding protein, has been identified to play an important role in cell proliferation and growth of normal tissues and tumor tissues, as well as tumor cell adhesion, apoptosis, migration, and invasion [28]. Studies have demonstrated that fibroblasts and smooth muscle cells from leiomyoma tissue not only increase the expression and secretion of growth factors including TGF-beta and IGF, but also up-regulate their corresponding receptors [29][30][31]. Notably, the overexpression of both proteins was associated with leiomyoma size and mass effect. Therefore, we strongly suspected that such changes in miR up-regulated TGFBR2 and IGF2BP1 in leiomyoma tissue and affected its growth.
Although we have investigated the pathogenic role of miRs in leiomyoma, the study has some limitations. First, the number of cases was relatively small to demonstrate the clinical significance other than leiomyoma size. Second, no direct interaction between miRs and protein expression was identified. Previous studies suggested that increased miRs such as miR-181a-5p, 28-3p and let-7 family may represent new therapeutic targets in various diseases [8,12,32]. Further studies are needed to validate the significance of the miRs as diagnostic or therapeutic targets.

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Taken together, the results demonstrated that miRs played a significant role in the pathogenesis of leiomyoma. Particularly, the five miRs including miR-181a-5p, 127-3p, 28-3p, 30b-5p and let-7c-5p, and their target molecules including TGFBR2 and IGF2BP1, were up-regulated in leiomyoma tissues. Additionally, their expression was associated with leiomyoma size and size effect. These findings are significant in understanding of miRs and their role as a potential indicator of leiomyoma development and disease progression.